HIV/AIDS is a major cause of global morbidity and mortality. Direct killing of infected CD4+ T cells and evasion of immune responses underlie the pathogenesis of AIDS. The membrane-associated viral Nef protein, which is critical for viral replication and virulence downregulates many immune cell receptors including CD4 and MHC class I. In the previous reporting period(s), we showed that both the recycling cell surface receptor and the nascent receptor in transit to the plasma membrane were susceptible to intra-cellular retention and degradation by HIV-1 Nef (J Cell Biology, 163, 1281, 2003 and J Biol Chem. 280, 7413, 2005). Nef is thought to mediate immune evasion and promote viral persistence in part by downregulating major histocompatibility complex class I protein (MHC-I or HLA-I) from the cell surface. Two different models have been proposed to explain this phenomenon: 1) stimulation of MHC-I retrograde trafficking from and aberrant recycling to plasma membrane versus 2) inhibition of anterograde trafficking of newly synthesized HLA-I from the ER to the plasma membrane. We show here that Nef simultaneously uses both mechanisms to downregulate HLA- I in PBMCs or HeLa cells. Consistent with this, we found using Fluorescence Correlation Spectroscopy that a third of diffusing HLA-I at the ER, GOLGI/TGN and the plasma membrane (PM) was associated with Nef. The binding of Nef was similarly avid for native HLA-I and recombinant HLA-I A2 at the PM. Nef binding to HLA-I at the PM was sensitive to specific inhibition of endocytosis. It was also attenuated by cyclodextrin disruption of PM lipid micro-domain architecture, a change, which also retarded lateral diffusion and induced large clusters of HLA-I. In all, our data support a model for Nef downregulation of HLA-I that involves both major trafficking itineraries and persistent protein-protein interactions throughout the cell. (J Biol Chem In Press, 2010) Chemokine receptors constitute a sub-class in GPCR (the G-protein coupled receptors) superfamily, which is phylogenetically conserved from yeast to primates and regulate diverse biological processes besides leukocyte chemotaxis. In previous years a) we identified the structural requirements of chemokine receptors for the biological function and HIV usage (J Biol Chem. 276, 40133, 2001);b) investigated how naturally occurring CCR5 mutants impaired for surface expression impact the physiological function of the wt receptor and its HIV usage in heterozygotes (J Biol Chem. 277, 2287, 2002);c) addressed the mechanistic differences between the CCR5 and CXCR4 receptors in the agonist-driven receptor signaling, and trafficking (Mol Biol of Cell 14, 3305, 2003), d) showed that in primary leukocytes, agonist driven cell migration and receptor internalization are separable processes (J. Biol Chem. 279, 24372, 2003);and e) showed that cholesterol was absolutely required for chemotaxis and associated spatial polarization of chemotactic mediators, but not for endocytosis of agonist-occupied receptors and associated signaling (J Biomed Sci 15, 441, 2008). Human chemokine and chemokine receptor dysfunction are associated with numerous acute and chronic diseases such as asthma and arthritis and infectious diseases including AIDS and TB. Our understanding of this feedback controlled signaling system in health and disease will enable us to understand viral pathogenesis and help us develop novel therapeutic interventions. HIV-1, HIV-2, and SIV Nef alleles are known to downmodulate several chemokine receptors. We show that Nef downmodulates some chemokine receptors to a variable extent based on the cell type, receptor, and primary cell donors. Of all the chemokine receptors examined, CXCR4 in different primary cells and tumor cell lines and CXCR1 and CXCR2 in certain cell lines were downregulated by different HIV and SIV Nef alleles in the context DNA transfection, single-cycle HIV infection or Nef protein transduction, although the downregulation was quite modest compared to that of CD4. We also show that both naturally truncated mutants of CXCR4 as in WHIMS or engineered truncated variants of CXCR1, CXCR2 and CXCR4 (which lack classical motifs for endocytosis) are downregulated more profoundly than wt receptors. Nef induced ubiquitination of CXCR4 through recruitment of E3 ligase, AIP4 followed by a dynamin dependent endocytosis into MVBs for proteolyis. These mechanisms were confirmed by interaction between Nef, AIP4 and CXCR4 and by reversal of Nef effect through siRNA knockdown of AIP4 or by a small molecular weight inhibitor of dynamin. Nef has been shown to inhibit agonist driven chemotaxis through CXCR4 and CCR5 receptors by subverting chemotactic signaling at multiple steps. We show that although Nef induced only a slight reduction of CXCR4 at the cell surface, the residual receptor was profoundly impaired for chemotaxis and ligand mediated receptor internalization. The chemotactic defect resulted from inhibition of early signaling steps such as dissociation of G-protein subunits, intracellular Ca++ flux, polarized accumulation of pAKT, F-actin polymers and other signaling molecules. These defects were a direct result of a specific proteolysis of Gai2 GTPase subunit but not of other G&#945;i isoforms or family members. Loss of G&#945;i2 subunit was profound and was comparable to that of Nef induced CD4 degradation and was demonstrable in many cell types. Reconstitution with exogenous G&#945;i2 or other isoforms did not significantly rectify the chemotaxis defect. Unlike Nef induced loss, siRNA mediated G&#945;i2 KD did not diminish CXCL12 dependent signaling or alter CXCR4 expression. By degrading one specific Gai subunit, Nef might induce leukocyte migratory and homing defects in AIDS pathology. The members of the Toll-like Receptor (TLR) family play an important role in innate immunity. Of the 11 TLRs that have been identified, TLR1/TLR6, TLR2 and TLR4 form homo- or hetero-dimers, which can recognize various bacterial lipopeptides. These TLRs on the cell surface of leukocytes regulate chemotaxis, phagocytosis, and immune activation. TLR ligand LPS at 0.02 ng/ml over 15 min inhibited chemotactic response of CCR2, CXCR4 and FPR in monocytes, but not in PMNs, T cells or myelomonocytic cell lines. LPS treatment abolished polarized accumulation of F-actin and PIP3 in response to chemokine stimulation in monocytes but not in PMNs. TLR stimulation did not alter initial G-protein dependent signaling or the kinetics of endocytosis of occupied chemoattractant receptors. Chemotaxis inhibition following short-term LPS treatment didnt result from downmodulation of signaling receptors or from inordinate increase in adhesion through integrin activation. However, chemokine stimulation of LPS treated monocytes displayed circumferential accumulation of activated integrin and F-actin polymers resulting from global recruitment of EPAC leading to unpolarized activation of Rap1.